Post-translational modifications control JARID enzyme activity during DNA damage

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Close to 75% of all non-small cell lung cancer (NSCLC) patients receive radiation therapy at some point in their treatment regimen whether with curative or palliative intent. Radiation, which is currently given in a highly localized fashion, causes massive DNA damage leading to DSBs and cancer cell death, yet on its own is not yet curative. Indeed, while some cancers are intrinsically resistant to IR, others acquire resistance by upregulating DNA repair pathways. Given modern advances in the targeted administration of radiotherapy, a paradigm shift leading to curing NSCLC or other tumors could occur if tumor tissue could be globally sensitized to radiation therapy. The epigenetic susceptibilities we propose to investigate here, may be the key. The oncogenic JARID/KDM5 histone demethylase subfamily of Jumonji enzymes, which are overexpressed in multiple malignancies, have recently defined roles in the DNA damage pathway: they mediate DNA repair by erasing trimethyl marks on active chromatin harboring H3K4me3 marks, thus stopping transcription and facilitating the recruitment of both homologous recombination and non-homologous end joining repair factors. The novel concept we propose here is that JARID enzymes must be modified post-translationally upon DNA damage, likely by radiation-activated ATM or ATR kinases, to enhance their histone demethylating activity on active chromatin, thus providing a mechanism to stop transcription and recruit repair factors to these sites, for cancer-cell survival. If true, this novel oncogenic activity of JARID enzymes would have significant implications for developing new approaches to sensitize lung tumors to ionizing radiation (IR), by selectively inhibiting the enhanced demethylase activity of JARID enzymes on chromatin. Our specific aims are to: 1.Determine if JARID enzymes are substrates of ATM kinases during the DNA damage response to IR: we will determine by mass spectrometry if JARID1B is phosphorylated by ATM/ATR kinases during the DNA damage response. We will map the site of phosphorylation and will mutate it to alanine (loss of function) or glutamic or aspartic acid (gain of function) to determine the impact on DNA repair dynamics, using isogenic cells lines expressing endogenous wt JARID1B (as controls) or null for JARID1B (knock out cells available). 2.Define how oncogenic JARID enzyme activity is modulated by post-translational modifications: we will measure the histone demethylase activity of unphosphorylated vs phosphorylated JARID1B enzyme in vitro and in cells. We will also determine the genomic sites JARID1B associates with in control vs. in cells undergoing DNA damage, and define if JARID1B recruitment to DSBs is dependent on phosphorylation. Our study will thus have wide impact to lung cancer patients by providing the molecular and mechanistic foundation for hypersensitizing tumors to radiation by using Jumonji inhibitors to curtail DNA repair through blocking the histone signals that trigger this repair.